Diagnosis and Treatment Monitoring of a Patient with Gastrointestinal Stromal Tumor by Next‐Generation Sequencing and Droplet Digital Polymerase Chain Reaction Assay of a PDGFRA Mutation in Plasma‐Derived Cell‐Free Tumor DNA

This article presents a case that highlights the use of liquid biopsies for treatment response monitoring, showing evidence of the promise of the application of plasma‐based mutation analysis in the diagnostic and response monitoring setting.

Abstract

In patients with a suspected malignancy, standard‐of care management currently includes histopathologic examination and analysis of tumor‐specific molecular abnormalities. Herein, we present a 77‐year‐old patient with an abdominal mass suspected to be a gastrointestinal stromal tumor (GIST) but without the possibility to collect a tumor biopsy. Cell‐free DNA extracted from a blood sample was analyzed for the presence of mutations in GIST‐specific genes using next generation sequencing. Furthermore, liquid biopsies were used to monitor the levels of mutant DNA copies during treatment with a tumor‐specific mutation droplet digital PCR assay that correlated with the clinical and radiological response. Blood‐based testing is a good alternative for biopsy‐based testing. However, it should only be applied when biopsies are not available or possible to obtain because overall, in only 50%–85% of the cell‐free plasma samples is the known tumor mutation detected.

In patients with a suspected malignancy, standard‐of‐care management currently includes histopathologic examination and analysis of tumor‐specific molecular abnormalities. For malignancies that are characterized by known serum tumor markers (i.e., prostate‐specific antigen in prostate cancer, carcinoembryonic antigen in colorectal cancer), analysis of blood could be indicative for diagnosis, treatment response, and detection of recurrence [1], [2]. Unfortunately, such biomarkers are not available for all malignancies.

Herein, we present a patient with an abdominal mass suspected to be a gastrointestinal stromal tumor (GIST) but without the possibility of collecting a tumor biopsy. Because GISTs are characterized by the presence of activating mutations in the tyrosine kinase domain of the KIT or PDGFRα gene in more than 80% of the cases [3], cell‐free DNA (cfDNA) extracted from a blood sample was analyzed for the presence of mutations in these genes using next‐generation sequencing (NGS). Liquid biopsies were used to monitor the levels of mutant DNA copies during treatment with a tumor‐specific mutation droplet digital polymerase chain reaction assay (ddPCR) to predict clinical response.

A 77‐year‐old woman presented at the hospital with pain and a palpable mass in the abdominal region; she also had anemia (Hb 5.4 mmol/l) for which she received a blood transfusion. A computed tomography (CT) scan showed a mass of 14 × 12 × 16 cm with loco‐regional tumor depositions. The primary mass was suspect to be a GIST (Fig. 1A). The largest lesion seemed to originate from the stomach, and additional liver and intra‐abdominal metastases were seen. Gastroscopy showed, besides chronic gastritis, no abnormalities. A radiologic biopsy of the mass was planned, but the patient deteriorated and a new CT scan showed massive pulmonary embolism, warranting therapeutic anticoagulation. The planned biopsy had to be canceled because of the high risk of bleeding.

Figure 1.

Computed tomography (CT) and positron emission tomography (PET) scans. (A): CT scan at the moment of presentations. An abdominal tumor of 11 × 14 cm with solid and necrotic parts is seen, suspected for gastrointestinal stromal tumor. (B): CT scan after 3 months of treatment. A large homogeneous cyst of approximately 20 cm is seen with almost no active tumor parts. (C): PET‐CT scan before start of treatment, uptake of fluorodeoxyglucose is clearly seen in the tumor. (D): PET‐CT scan 1 week after start of treatment with imatinib 400 mg; almost no uptake is present anymore in the primary tumor, and physiological uptake at both kidneys is still present.

The patient gave permission to participate in a study to perform mutation analysis on cell‐free DNA extracted from plasma of patients with advanced GIST (KWF research grant RUG 2013‐6355, ClinicalTrials.gov NCT02331914). cfDNA was extracted and sequenced using NGS as reported previously [4]. The analysis revealed a mutation in PDGFRα (NM_006206.5: c.2524_2532del; p.D842_M844del), with a variant allelic frequency (VAF) of 4%. This PDGFRα c.2524_2532del; p.D842_M844del mutation was reported in GIST once in the COSMIC database [5]. Validation with an in house designed mutation‐specific ddPCR assay confirmed the presence of this mutation in cfDNA with a VAF of 2.9%. Imatinib sensitivity of a patient with this mutation was reported in 2015 [6]. Standard first‐line treatment with the tyrosine kinase inhibitor imatinib 400 mg was initiated. Shortly after initiation of therapy, the patient needed fewer blood transfusions and was able to leave the hospital. Positron emission tomography (PET) and CT scans performed before and 1 week after start of treatment showed a decrease in metabolic activity (Fig. 1C, 1D). A CT scan performed 3 months after start of treatment showed a large cystic lesion with only minimal active tumor lesions, indicating response to treatment (Fig. 1B). During treatment, the mutation‐specific ddPCR assay was used at baseline and 1, 3, 4, 8, and 16 weeks after start of targeted therapy for cfDNA analysis. This showed a gradual decrease in mutant copies per mL of plasma, suggesting treatment response (Fig. 2). Because the cfDNA results based on levels of mutants per mL of plasma are in very good agreement with the observed response as determined with PET/CT scanning, our data suggest that the minimal invasive cfDNA assay might be an interesting alternative to monitor response. With an average turnaround time of 1–2 and 5–7 days, respectively, for ddPCR and NGS in a diagnostic setting, it is applicable in daily clinical practice [7].

Figure 2.

Detection of mutant copies per mL using droplet digital polymerase chain reaction (ddPCR) analysis to detect the PDGFRα c.2524_2532del as measured before and during treatment. In the weeks after start of treatment, the mutant copies per mL decreased further; this is in good concordance with the tumor response as seen by positron emission tomography and computed tomography scan after 3 months, in which no active tumor lesions are seen (Fig. 1C, 1D). Procedures for DNA extraction and ddPCR analysis were reported previously [4], [6]. Upon request, details on primers and probes could be provided.

The course of the mutant allelic fraction in the cfDNA as measured with ddPCR correlates with the clinical and radiological response; however, after 5 days of treatment, a flare is detected in the cfDNA mutational level. We observed a similar flare 1–2 weeks after start with imatinib in 5 of 11 patients with GIST with a detectable KIT‐exon11 mutation and/or deletion in the baseline sample [8]. We hypothesize that this increase is caused by massive DNA shed by dying tumor cells caused by the initiation of treatment. This phenomenon has been described earlier in patients with colorectal cancer (CRC) shortly after start of treatment and could be used as a marker for early response [9].

Blood‐based testing is a good alternative for biopsy‐based testing. However, it should only be applied when biopsies are not available or not possible to obtain because overall in only 50%–85% of the cell‐free plasma samples (depending on disease status) the known tumor mutation is detected [10]. Recently, we reported on the feasibility of detecting KIT/PDGFRα mutations in baseline‐pretreatment plasma samples in a series of patients with GIST with parallel analysis of a pretreatment biopsy with NGS. This study showed that the sensitivity of detecting a relevant KIT mutation in baseline plasma is ~90% in metastasized GIST [8]. Furthermore, a tissue biopsy provides important information on histology, diagnostic, and tumor‐specific immunophenotypic markers and mostly a higher amount of neoplastic cells for molecular testing.

Detection of mutations in patients with GIST using liquid biopsy was reported previously [11], [12], [13], [14], [15]. These studies used sequencing as well as polymerase chain reaction‐based techniques for detection of mutations in tumor and used the known tumor mutation for monitoring therapy in plasma.

As the circulating tumor DNA (ctDNA) in plasma might originate from different lesions (the primary tumor and its metastases), liquid biopsies can provide information regarding the spatial and temporal heterogeneity of tumors. Monitoring the mutational levels in plasma in time can guide personalized treatment and could possibly improve treatment outcome.

The use of liquid biopsies has been extensively validated in non‐small cell lung cancer, melanoma, and CRC [16], [17], [18]. Practically all applications of liquid biopsy are regarding the monitoring of treatment response to (targeted) treatment and detection of secondary treatment resistant mutations. In 2016, the Food and Drug Administration approved ctDNA analysis in patients with lung cancer for initial genotyping of tumors when insufficient tissue is available for molecular characterization. This is exemplary of a shift in the use of liquid biopsies to broader applications, also in a diagnostic setting.

This case highlights the use of liquid biopsies for treatment response monitoring. The application of plasma‐based mutation analysis in a diagnostic and response monitoring setting seems promising and has several advantages when compared with traditional methods. However, more and larger prospective studies are needed to confirm the use and justify the implementation of this technique into daily practice.

Disclosures

Ed Schuuring: Biocartis, Bristol‐Myers Squibb, Bio‐Rad, Roche, Cancer‐ID (RF, institutional). The other authors indicated no financial relationships.

(C/A) Consulting/advisory relationship; (RF) Research funding; (E) Employment; (ET) Expert testimony; (H) Honoraria received; (OI) Ownership interests; (IP) Intellectual property rights/inventor/patent holder; (SAB) Scientific advisory board